The mechanism of target cell lysis by LGL tumor granule cytolysin has been studied by several different approaches. Since previous results had suggested a complement-like protein insertion and pore formation, we first showed that liposomes were targets for the cytolysin in a rapid, calcium-dependent reaction leading to carboxyfluorescein release. Cylindrical pore-like structures were shown to be inserted into liposomes which also displayed penetration of negative stain. Similarly, electrical measurements on artificial membranes showed that cytolysin induced a calcium-dependent ionic permeability increase which was highly voltage dependent and identical in properties to that previously described with lymphocytes in an ADCC model. Confirming the protein insertion model, it was shown that liposomes and lipoproteins were highly inhibitory to the lytic activity of cytolysin. Antibodies raised against purified granules specifically stained LGL granules in fluorescent microscopy, and F(ab')2 fragments specifically neutralized cytolytin activity as well as ADCC and NK activity. These results strongly suggest that a granule component is involved in the cell-mediated cytotoxic process. Additional studies were directed at the differences in sensitivity to the cytolysin of various nucleated target cells. The resistant cells are capable of inactivating cytolysin in a calcium-independent process that is not understood, but which appears to account for the difference in sensitivity to the cytolysin. Cytolysin has been purified by solubilization in 2M NaCl and gel filtration on ACA 54, where it elutes at the position of a 60 kd. protein. Further purification can be accomplished by DEAE-HPLC, but the preparation still contains several bands on SDS gels.